PCR-based identification of Trypanosoma lewisi and Trypanosoma musculi using maxicircle kinetoplast DNA
PCR-based identification of Trypanosoma lewisi and Trypanosoma musculi using maxicircle kinetoplast DNA
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DOI：
10.1016/j.actatropica.2017.04.007
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发表时间：
2017-07-01
期刊：
影响因子：
2.7
通讯作者：
Lai, De-Hua
中科院分区：
文献类型：
--
作者：
Hong, Xiao-Kun;Zhang, Xuan;Lai, De-Hua
路易斯锥虫由鼠蚤传播，是一种广泛传播的病原体，专门针对老鼠，有人类感染病例的记录。其密切相关的物种是全球分布的锥虫，通过摄入受感染的跳蚤在小鼠之间传播。这些锥虫具有相似的形态，因此很难通过显微镜区分它们。在本研究中，我们开发了一种快速、灵敏且可靠的 PCR 方法来诊断 T. lewisi 和 T. musculi。路易斯锥虫特异性扩增子不是由其他锥虫产生的，例如肌锥虫、布氏锥虫复合体或克氏锥虫，亚马逊利什曼原虫的外类群也不产生。三对 T. lewisi 特异性引物的检测限分别为 50 ng、1 ng 和 10 ng 总 DNA。为 T. musculi 引物设计的引物明确表明，扩增子严格存在于 T. musculi 中，其检测限分别为 10 ng 和 1 ng 总 DNA。为了简化检测过程，我们成功地将我们的方法直接应用于尾血样本，无需复杂的 DNA 纯化。总之，使用我们的引物进行的 PCR 可能是一种高度敏感的特异性方案，用于检测并将 T. lewisi 和 T. musculi 与其他锥虫区分开来。
Trypanosoma lewisi, transmitted by rat fleas, is a widespread pathogen specific to rats with records of human infection cases. Its closely related species with global distribution, Trypanosoma musculi, is transmitted between mice by ingestion of infected fleas. These trypanosomes are of similar morphology, making it difficult to distinguish them by microscopy. In this study, we have developed a rapid, sensitive and reliable PCR method for the diagnosis of T. lewisi and T. musculi. The T. lewisi-specific amplicons were not produced by other Trypanosoma, such as T. musculi, T. brucei complex or T. cruzi, neither by an outgroup of Leishmania amazonensis. The detection limits of the three pairs of T. lewisi-specific primers were 50 ng, 1 ng and 10 ng of total DNA, respectively. The primers designed for T. musculi primers showed specifically that amplicon strictly in T. musculi and their detection limits were 10 ng and 1 ng of total DNA. To simplify the detection process, we managed to apply our method directly on tail blood samples without complicated DNA purification. In conclusion, PCR with our primers could be a highly sensitive, specific protocol to detect and distinguish T. lewisi and T. musculi from other trypanosomes.